CN109536574A - A kind of colorimetric method of simple detection fibrin ferment - Google Patents

A kind of colorimetric method of simple detection fibrin ferment Download PDF

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CN109536574A
CN109536574A CN201811249045.7A CN201811249045A CN109536574A CN 109536574 A CN109536574 A CN 109536574A CN 201811249045 A CN201811249045 A CN 201811249045A CN 109536574 A CN109536574 A CN 109536574A
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fibrin ferment
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thrombin
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王颖
李风亭
张冰如
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Tongji University
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Abstract

The present invention provides a kind of colorimetric method of simple detection fibrin ferment, there is the characteristic of Mimetic Peroxidase in acid condition using metal organic framework compound Fe-MIL-88A, the fibrin ferment tetramer is generated by Thrombin aptamer and blood coagulation enzyme reaction, then again with Fe-MIL-88A and 3,3 ', 5,5 '-tetramethyl benzidine (TMB)-H2O2System carries out catalytic oxidation, using ultraviolet spectrophotometry and colorimetric method for determining concentration of thrombin.The present invention acts on the specific binding of substrate using the mimetic enzyme catalysis performance and aptamer of Fe-MIL-88A, can sensitive, accurate, specific and visual detection fibrin ferment.

Description

A kind of colorimetric method of simple detection fibrin ferment
Technical field
The present invention relates to technical field of analysis and detection, more particularly, to a kind of colorimetric method of simple detection fibrin ferment, specifically For the colorimetric method that metal organic framework compound material Fe-MIL-88A is used to detect fibrin ferment.
Background technique
Fibrin ferment (thrombin) is a kind of very important serine protease in body blood coagulation system, it is by 308 Amino acid residue composition, molecular weight 37kD.Fibrin ferment has in blood coagulation system promotees solidifying and anticoagulant dual function, in inflammation tune Section, angiogenesis play an important role in tissue repair and oncobiology, also have in central nervous system pathology and Physiological action.Fibrin ferment is considered as the important measurement index and some diseases diagnosis (such as Lung metastases and cartilage of clotting mechanism Synovial membrane inflammation) biological marker.Therefore, fibrin ferment detects the monitoring to the early diagnosis of clinical disease, disease, curative effect With assessment etc. all have very important significance.
The method of detection fibrin ferment mainly has red, orange, green, blue, yellow (ROGBY), fluorescence method immunoassay and aptamer method at present.By In aptamer sensor due to having many advantages, such as that combining target species are wide, affinity is high, high specificity, at low cost, It is widely used in analysis detection field.Wherein, colorimetric method is to be measured to determine by comparing measurement coloring matter solution colour depth The method of constituent content, may be implemented Visual retrieval, show greatly advantage in fibrin ferment detection application aspect.
Patent CN201510073478.1 provides a kind of based on gold-hydrogen peroxide variable color system and rolling circle amplification phase In conjunction with blood coagulation enzyme assay method.In the presence of hydrogen peroxide, gold ion is reduced into nanogold, solution presents red;When solidifying Hemase aptamer and annular template interconnect, and carry out rolling circle amplification and generate DNA enzymatic.Since DNA enzymatic can be catalyzed peroxidating Hydrogen decomposes, and reduces concentration of hydrogen peroxide, forms the nanogold of reunion, and blue is presented in solution.The rolling circle amplification that the invention uses Technology, complex steps, reaction time are long.
Patent CN201180059028.7 provides a kind of for comprising Antithrombin III (AT-III) and fibrin ferment The method that in vitroimmunoassay is carried out to fibrin ferment in sample.First by the substrate binding site of fibrin ferment and identification fibrin ferment Small molecule contact;Secondly, fibrin ferment is contacted with Thrombin specificity antibody.Horizontal by measurement binding antibody realizes blood coagulation The detection of enzyme.This kind of method, cumbersome, required biological reagent is more, and condition of storage is harsh.
Metal organic framework compound (Metal-Organic Framework, MOF) is one kind developed in recent years The polymer with infinite network structure connected by inorganic metal ion and organic ligand by unlimited coordination mode is more Porous materials.Since metal organic framework compound has many advantages, such as porosity, hole controllability, is easy to functionalization, in molecule The fields such as detection show good application prospect (Li Song, the design synthesis and visualization inspection of metal-organic framework crystalline material Survey metal ion probe research [D], South China Science & Engineering University, 2015).
Studies have shown that part MOF material has Mimetic enzyme of peroxidase catalytic performance.Studies have found that Fe-MIL-53 With Mimetic Peroxidase catalytic performance, and be used for hydrogen peroxide detection, have good detection effect (Ai L, Li L, Zhang C, Fu J, Jiang J.MIL-53 (Fe): a metal-organic framework with intrinsic peroxidase-like catalytic activity for colorimetric biosensing[J] .Chemistry.2013,19 (45): 15105-8.).In addition scholar has studied Fe-MIL-88NH2Peroxidase work Property and be applied to glucose detection (Liu YL, Zhao XJ, Yang XX, Li YF.A nanosized metal-organic framework of Fe-MIL-88NH(2)as a novel peroxidase mimic used for colorimetric Detection of glucose [J] .The Analyst.2013,138 (16): 4526-31.).
Aptamer is that the energy specificity screened in vitro by index concentration Fas lignand system evolution technology is known A kind of single-stranded oligonucleotide of other target molecule.Aptamer is with specific bond object type is wide, affinity is high, specific By force, the advantages that easily modifying, is widely used in analysis detection field (Zheng Jing, He Pingang, the aptamer biosensor of square Yu [J] chemical progress, 2009,04:732-738.).But nucleotide aptamer is mainly used for detecting biological micromolecule at present, thinks The difficult of detection also many technical aspects for carrying out large biological molecule needs to overcome.
Summary of the invention
In view of the above-mentioned problems existing in the prior art, this application provides a kind of colorimetric methods of simple detection fibrin ferment.This Invention uses sensitive Colorimetric System, and the blue solution generated using Fe-MIL-88A catalyzing hydrogen peroxide oxidation TMB is directly sentenced The presence of disconnected fibrin ferment;It is simple and easy it is not necessary that aptamer is marked in advance, it is not necessarily to complex instrument equipment.
Technical scheme is as follows:
A method of detection fibrin ferment, which comprises the steps of:
(1) sample to be detected and Thrombin aptamer are mixed to get the fibrin ferment tetramer;
(2) the fibrin ferment tetramer is mixed with Fe-MIL-88A solution;
(3) 3,3 ', 5 are added in the mixed liquor obtained to step (2), 5 '-tetramethyl benzidines, that is, TMB, H2O2Solution and vinegar Sour sodium buffer, carries out catalysis reaction;
(4) sulfuric acid solution is added, absorbance is surveyed using ultraviolet specrophotometer, by test result and standard fibrin ferment curve Comparison, obtains the concentration of thrombin of sample to be tested.
Wherein, the concrete operation method of step (1) are as follows:
The 5 '-TCT CTC AGT CCG TGG TAG GGC AGG of Thrombin aptamer for being 1 μM by 100 μ L concentration TTG GGG TGA CT-3 ' reacts 1 hour with 50 μ L samples to be detected in D-PBS buffer at 25 DEG C, obtains blood coagulation The enzyme tetramer;
The D-PBS pH of buffer is 7.4, and group becomes potassium chloride 2.67mmol/L;Dipotassium hydrogen phosphate 8.1mmol/L;Phosphorus Acid dihydride sodium 1.47mmol/L;Sodium chloride 138mmol/L;Magnesium chloride 5mmol/L.
The concrete operation method of step (2) are as follows:
The Fe-MIL-88A solution that 10 μ L concentration are 0.4mg/mL, concussion reaction 3 are added in the fibrin ferment tetramer Minute.
The concrete operation method of step (3) are as follows:
The H that TMB, 10 μ L concentration that 100 μ L concentration are 10mM are 10mM is added in the mixed liquor obtained to step (2)2O2It is molten The HAc-NaAc buffer of the pH 3.0 of liquid and 730 μ L react 20 minutes at 40 DEG C.
The concrete operation method of step (4) are as follows:
The H that 50 μ L concentration are 2mM is added into mixed liquor after reaction2SO4Solution reads absorbance at 451nm Value.
Step (4) the standard fibrin ferment curve, which refers to, carries out step (1)~(3) for the thrombin solution of normal concentration After processing, sulfuric acid solution is added, absorbance is surveyed using ultraviolet specrophotometer, using corresponding concentration of thrombin as abscissa, with this Concentration thrombin solution absorbance at ultra-violet absorption spectrum 451nm is the curve that ordinate is drawn.Standard curve is specifically drawn Method processed are as follows:
(1) synthesis of Fe-MIL-88A: Iron(III) chloride hexahydrate and fumaric acid are dissolved in ultrapure water first, then Entire homogeneous phase solution, which is transferred in reaction kettle, heats reaction.After being cooled to room temperature, reactant water and ethyl alcohol clean repeatedly, centrifugation Product Fe-MIL-88A powder is obtained after being dried overnight in a vacuum drying oven afterwards.It weighs the powder and is dissolved in ultrasound in ultrapure water Fe-MIL-88A solution is formed after mixing.
The ultrapure water refers to that the conductivity prepared with Milli-Q ultrapure water instrument is 18.2M Ω cm-1Pure water.
The concentration of the Fe-MIL-88A solution is 0.4mg/mL;Storage temperature: normal temperature storage.
(2) formation of the fibrin ferment tetramer: Thrombin aptamer and fibrin ferment are reacted in D-PBS solution, are formed Fibrin ferment tetramer solution, includes the following steps:
I, Thrombin aptamer freeze-dried powder is centrifuged 10 minutes under the conditions of 10000rmp, D-PBS buffer solution is added, It is configured to Thrombin aptamer titer.
The concentration of the Thrombin aptamer titer is 1 μM, condition of storage are as follows: storage temperature: 0-4 DEG C;When storage Between: within 1 month.
The D-PBS is achieved through the following technical solutions:
II, thrombin standard substance solution is weighed, with ultrapure water dissolution and constant volume, preparing standard solution.
The concentration of the thrombin standard solution is 2 μm of ol/L.
The condition of storage of the thrombin standard solution are as follows: storage temperature: 0-4 DEG C;Storage time: within 1 month.
III, the thrombin standard solution pipetted in the step II of different volumes respectively are divided with ultrapure water dilution and constant volume It is not formulated as a series of thrombin standard solution of various concentrations.
The standard solution should cover the concentration range of fibrin ferment in sample to be tested, can be i.e. with i.e. use.
The thrombin standard solution of Thrombin aptamer and various concentration is mixed, is reacted 1 hour at room temperature, is formed Fibrin ferment tetramer standard solution.
(3) TMB catalysis reaction: the fibrin ferment four for respectively generating Fe-MIL-88A solution in step (1), step (2) Aggressiveness standard solution carries out mixing and carries out catalysis reaction.
I, TMB standard substance is weighed, with ethanol solution dissolution and constant volume, configures TMB solution.
The concentration of the TMB solution is 10mmol/L.
The condition of storage of the TMB solution are as follows: storage temperature: 0-4 DEG C;Storage time: within 1 month.
II, H is pipetted2O2Solution, with ultrapure water dilution and constant volume, preparing standard solution.
The H2O2The concentration of standard solution is 10mmol/L.
The H2O2The condition of storage of standard solution are as follows: normal temperature storage.
III, acetic acid and acetic acid sodium crystal are weighed, with ultra-pure water solution and constant volume, configures sodium acetate buffer (pH= 3)。
IV, after being added after mixing Fe-MIL-88A and fibrin ferment tetramer standard solution, TMB, H are sequentially added2O2, vinegar Sour sodium buffer, 40 DEG C are reacted 20 minutes.
(4) detect: after reaction, into reaction solution be added sulfuric acid terminate liquid, after UV-1700 UV absorption be divided Photometric determination absorption spectrum.
The concentration of the sulfuric acid terminate liquid is 2mol/L.
(5) standard curve is drawn.
Corresponding standard working curve is drawn according to the ultra-violet absorption spectrum that step (4) obtains, calculates separately to obtain blood coagulation The regression equation of enzyme standard working curve.
In the standard curve, using at ultra-violet absorption spectrum 451nm absorbance as ordinate (Y-axis), corresponding blood coagulation Enzyme concentration is abscissa (X-axis).
The detection of ultraviolet specrophotometer carries out at room temperature.Spectral scan is opened from wave-length coverage from 400nm Begin to 800nm to terminate.
After obtaining above-mentioned standard curve, by the processing result and standard curve control of sample to be tested, fibrin ferment can be obtained Concentration.It can also be scanned without ultraviolet light spectrophotometer, but after terminating reaction, only compare by visually to test sample The color of product and thrombin standard sample judges the concentration of sample to be tested fibrin ferment by colorimetric method.
Metal organic framework compound material Fe-MIL-88A has Mimetic Peroxidase catalytic, can be catalyzed H2O2 TMB is aoxidized, solution becomes blue;After the completion of reaction, sulfuric acid terminate liquid is added, solution turns yellow.
The purpose of the present invention is to provide a kind of colorimetric methods of simple detection fibrin ferment, for accurate and reliable, operation to be simple Realize the detection of colorimetric method fibrin ferment in ground.To achieve the above object, the present invention is raw using Thrombin aptamer and blood coagulation enzyme reaction At the fibrin ferment tetramer, then with a kind of iron content organic framework compounds Fe-MIL-88A and TMB-H2O2Catalysis reaction is carried out, is used The concentration of ultraviolet spectrophotometer method measurement fibrin ferment.
By sample to be tested formed the fibrin ferment tetramer and with Fe-MIL-88A, TMB, H2O2After equal solution reactions, using purple The result of outer spectrophotometer detection need to obtain the concentration of thrombin number of sample to be tested with the data comparison of thrombin standard curve According to.
The present invention is beneficial to be had the technical effect that
The advantages of present invention combination metal organic framework compound mimetic enzyme catalysis performance and aptamer it is highly selective And specificity, establish a kind of visualization blood coagulation enzyme assay method simple, sensitive, specificity is high.The present invention is loaded using MOF Fibrin ferment aptamer forms void effect, realizes fibrin ferment detection, constructs point specifically by MOF and aptamer The duct of sub switch.The duct of molecular switch is opened through aptamer in the presence of no target, and small molecule TMB can be with Into duct, acts on and developing the color by MOF mimetic enzyme catalysis;In the presence of having target molecule, aptamer identifies molecule, forms four Aggressiveness, duct are closed, and small molecule TMB cannot enter duct, MOF enzymatic miopragia or disappearance, cannot be developed the color, thus real Now detect.How under suitable conditions technological difficulties therein are rapid build ducts, and it is tired that this is that the present invention overcomes technologies Difficult place.
The range of linearity that the present invention measures fibrin ferment is 20-80nmol/L, and detection is limited to 0.8nmol/L.Utilize different lifes The amino acid of object macromolecular and different isoelectric points shows selection of the MOF- aptamer sensor to fibrin ferment as interfering substance Property is good;Using fibrin ferment recovery testu in serum, the rate of recovery of fibrin ferment is measured between 99.3% to 100.4%, Relative standard deviation is between 0.3% to 1%.
Detailed description of the invention
Fig. 1 is the transmission electron microscope picture of Fe-MIL-88A;
Fig. 2 is the XRD spectrum of Fe-MIL-88A;
Fig. 3 is the ultraviolet spectra response diagram and calibration curve of fibrin ferment in detection method;
Fig. 4 is the changing rule of the thrombin solution color of various concentration;
Fig. 5 is the anti-interference schematic diagram of MOF- aptamer sensor;
Fig. 6 is the mimetic enzyme catalysis performance test result of Fe-MIL-88A described in comparative example 1: a) TMB;b)TMB+H2O2;c) TMB+Fe-MIL-88A;d)Fe-MIL-88A+H2O2+TMB;Interior illustration: the color change comparison diagram of solution, vial a are corresponding It is line a, it is other similar;
Fig. 7 is the condition optimizing result of Fe-MIL-88A mimetic enzyme catalysis described in comparative example 2: A) H2O2Concentration;B)Fe- MIL-88A concentration;C) pH and D) temperature.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with the drawings and specific embodiments The invention will be further described.It should be appreciated that preferred embodiments described herein is merely to illustrate and explain the present invention, and It is not used in the restriction present invention.
Metal organic framework compound Fe-MIL-88A, Thrombin aptamer and fibrin ferment is cleverly utilized in the present invention Interaction relationship between each substance designs a kind of colorimetric method that can be used in detecting thrombin amount.Fe-MIL-88A can In a certain amount of H2O2TMB colour developing is catalyzed under the conditions of existing, solution becomes blue from colourless.Under certain conditions, fibrin ferment core The capture prothrombin molecule of the sour aptamer property of can choose forms the fibrin ferment tetramer.The fibrin ferment tetramer is added to Fe-MIL- 88A and TMB-H2O2In system, under the action of electrostatic attraction, the fibrin ferment tetramer can be wrapped in around Fe-MIL-88A, be hindered Electron transmission between Fe-MIL-88A and TMB inhibits the progress of oxidation reaction, it is non-discolouring to show as solution to be measured.Pass through light splitting The highly sensitive specific detection to fibrin ferment may be implemented in photometry and colorimetric method.
Embodiment 1
(1) synthesis of Fe-MIL-88A
First by 5mM Iron(III) chloride hexahydrate (FeCl3·6H2O it) is dissolved in 25mL ultrapure water with 5mM fumaric acid, then Entire homogeneous phase solution, which is transferred in 60mL reaction kettle, is heated to 65 DEG C of reactions 12 hours.After being cooled to room temperature, reactant water and Ethyl alcohol cleans repeatedly, obtains product Fe-MIL-88A powder after being dried overnight in vacuum oven (being lower than 80 DEG C) after centrifugation End.It weighs the 4.0mg powder and is dissolved in 10.0mL ultrapure water formation 0.4mg/mL Fe-MIL-88A solution after ultrasonic mixing.
It will be seen from figure 1 that Fe-MIL-88A is a kind of fusiform crystal.Figure it is seen that Fe-MIL-88A has There is specific crystal structure.
(2) prepared by fibrin ferment MOF- aptamer mixture
100 μ L Thrombin aptamer, 5 '-TCT CTC AGT CCG TGG TAG GGC AGG TTG GGG TGA CT- 3 ' (concentration is 1 μM) and the fibrin ferment of 50 μ L various concentrations react 1 hour at room temperature.After reaction, to mixed liquor 10 μ LFe-MIL-88A (0.4mg/mL) of middle addition, 730 μ LHAc-NaAc buffers (pH 3.0), 100 μ L TMB solution (10mM, ethyl alcohol are solvent) and 10 μ L H2O2(10mM), 40 DEG C are reacted 20 minutes.50 μ L are added into mixed liquor after reaction H2SO4Absorption spectrum is measured in UV--1700 Ultravioblet spectrophotometer after (2M).
(3) Specification Curve of Increasing
When drawing standard curve, with ultrapure water dilution and constant volume concentration is the thrombin solution of 2 μm of ol/L, is formulated as respectively A series of thrombin standard solution of various concentrations.
The detection of ultraviolet specrophotometer carries out at room temperature.Spectral scan wavelength since 400nm to 800nm terminates.Fe-MIL-88A aoxidizes TMB reaction, and solution is made to become blue.The judgment basis of colorimetric method, i.e., according to solution indigo plant The depth of color judges the concentration range of fibrin ferment, and color is deeper, and concentration of thrombin is smaller.But blue solution is in ultraviolet light There is higher background value in spectrum, therefore, in UV spectrophotometer measuring method, after reaction, is added to blue solution H28O4(2M) is used as reaction terminating liquid.With the addition of sulfuric acid solution, solution is turned yellow from blue at once, is gone out at 451nm Existing characteristic absorption peak.
Using absorbance at ultra-violet absorption spectrum 451nm as ordinate (Y-axis), the concentration of corresponding thrombin standard solution is Abscissa (X-axis), draws corresponding standard working curve, and the regression equation of thrombin standard working curve is calculated.Fig. 3 is The ultraviolet spectra response diagram and calibration curve of fibrin ferment.
Under the influence of the fibrin ferment tetramer, Fe-MIL-88A is catalyzed H2O2Aoxidize TMB colour developing reaction and fibrin ferment it is dense Spend it is in a linear relationship, can establish accordingly visualization fibrin ferment determination method.Under optimum reaction condition, oxidation The concentration of absorbance and fibrin ferment of the TMB at 451nm has preferable linear relationship in a certain range.Its linear detection range For 10nM-80nM (R2=0.9906), detection is limited to 0.8nM, and (LOD=3SD/S, wherein SD is the deviation of blank sample, and S is mark The numerical value of the slope of directrix curve, each point in standard curve is the average value of 3 measurement results).
Fig. 4 is color camera corresponding to the fibrin ferment of system of determination various concentration concentration.With the increasing of concentration of thrombin Greatly, the blue of solution is more and more shallow.
(4) colorimetric determination
Sample to be tested is handled according to above-mentioned steps (1), the method for (2), then uses Ultravioblet spectrophotometer Absorbance data is detected, is compared with the thrombin standard solution colour (i.e. Fig. 4) after standard curve (i.e. Fig. 3) or reason, Obtain the concentration of thrombin of sample to be tested.
Embodiment 2: anti-interference test
To probe into the method for the present invention to the anti-interference ability of the amino acid of other potential interference protein and different isoelectric points, We are to bovine serum albumin (BSA), immunoglobulin G (IgG), lysozyme (lysozyme), lysine (lysine), arginine (arginine), glycine (glycine), sodium glutamate (L-sodium glutamate, LSG) have carried out parallel disturbed test Experiment.From fig. 5, it can be seen that only existing for the fibrin ferment under the conditions of, absorbance reduced rate significantly increases, and solution almost becomes It is colourless.Under the conditions of existing for other high molecular weight proteins or the amino acid of various isoelectric points, the analogue enztme of Fe-MIL-88A is urged The property changed does not receive apparent inhibiting effect, and solution is still blue.The experimental results showed that detection method of the invention is with good Good interference free performance and fibrin ferment evident characteristics.
Embodiment 3: mark-on is tested in serum
It uses and the fibrin ferment of standard is added in Standard entertion normal direction cow's serum to be configured to the blood serum sample of various concentration.Blood It is to remove left next blood plasma after fibrin after blood clotting clearly.The chemical component of serum is similar with blood plasma, but does not contain Blood coagulating protein.Each group of mark-on experiment all repeats 3 groups of parallel laboratory tests, and the final data in table 1 is being averaged for 3 groups of parallel laboratory tests Value.The result shows that the rate of recovery of fibrin ferment is between 99.3% to 100.4%, relative standard deviation between 0.3% to 1%, Accuracy and repeatability of the present invention are preferable, can satisfy quantitative needs.
1 serum mark-on of table tests the thrombin samples rate of recovery
Comparative example 1:
By being catalyzed H2O2Oxidation TMB reacts to verify the mimetic enzyme catalysis performance of Fe-MIL-88A.Reaction condition: TMB, 1mM;H2O2, 100 μM;Fe-MIL-88A, 4 μ g/mL;HAc-NaAc(pH 3.0);40 DEG C are reacted 20 minutes.
As a result as shown in Fig. 6 line a and line b, in only H2O2Suction under conditions of addition, at 652nm ultra-violet absorption spectrum Peak is received almost without what variation, TMB solution is still that colourless (Fig. 6 bottle b) shows TMB almost without aoxidizing.Once Fe- MIL-88A is added to TMB-H2O2In system, the absorption peak at 652nm ultra-violet absorption spectrum significantly increases (Fig. 6 line d), solution (Fig. 6 bottle d) shows that Fe-MIL-88A can be very good oxidation matrix TMB to the apparent blue that becomes.
Comparative example 2:
Same horseradish peroxidase (HRP) is similar, the mimetic enzyme catalysis activity of Fe-MIL88A be heated temperature, pH value, H2O2The influence of concentration and catalytic materials concentration.Therefore, this experiment has studied temperature, pH value, H respectively2O2Concentration and Fe-MIL- Influence of the 88A concentration to Fe-MIL-88A mimetic enzyme catalysis.
Firstly, this experimental study Fe-MIL-88A is in different H2O2The catalytic activity of (100 μM of -400mM) under concentration.It will The H of various concentration2O2Solution is added to containing 0.2mM TMB, 0.04mg/mL Fe-MIL-88A, sodium-acetate buffer (pH 3.0) it in mixed liquor, is reacted 20 minutes in 40 DEG C of water-baths.The H of high concentration as can be seen from Figure 7A2O2Can have one to catalysis Fixed inhibiting effect, but the H of Fe-MIL-88A2O2Tolerable concentration is higher than HRP, shows the H in high concentration2O2Under the conditions of, Fe-MIL-88A ratio HRP is more stable.
Then, influence of the concentration of this experimental study Fe-MIL-88A to catalysis reaction.The Fe- of 0.01-0.05mg/mL MIL-88A is added to containing 0.2mM TMB, 400 μM of H2O2, in the mixed liquor of sodium-acetate buffer (pH 3.0), in 40 DEG C of water 20min is reacted in bath.When Fe-MIL-88A amount is 0.04mg/mL, catalytic activity reaches maximum (Fig. 7 B).
Then, influence of this experimental study pH value to catalysis reaction.0.2mM TMB, 400 μM of H2O2, 0.04mg/mL Fe-MIL-88A is added in the sodium-acetate buffer of different pH value (2.5-6), is reacted 20 minutes in 40 DEG C of water-baths.Experiment Show that Fe-MIL-88A can guarantee good catalytic effect (Fig. 7 C) in the range of pH value is 2.5-4.
Finally, 0.2mM TMB, 400 μM, 0.04mg/mL Fe-MIL-88A and sodium-acetate buffer (pH 3.0) are in difference It is heated 20 minutes in the water-bath of temperature, to detect influence (Fig. 7 D) of the temperature to catalysis reaction.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as At all equivalent modifications or change, should be covered by the claims of the present invention.

Claims (6)

1. a kind of method for detecting fibrin ferment, which comprises the steps of:
(1) sample to be detected and Thrombin aptamer are mixed to get the fibrin ferment tetramer;
(2) the fibrin ferment tetramer is mixed with Fe-MIL-88A solution;
(3) 3,3 ', 5 are added in the mixed liquor obtained to step (2), 5 '-tetramethyl benzidines, that is, TMB, H2O2Solution and sodium acetate Buffer carries out catalysis reaction;
(4) sulfuric acid solution is added, absorbance is surveyed using ultraviolet specrophotometer, by test result and standard fibrin ferment curve pair Than obtaining the concentration of thrombin of sample to be tested.
2. the method according to claim 1, wherein the concrete operation method of step (1) are as follows:
The 5 '-TCT CTC AGT CCG TGG TAG GGC AGG TTG of Thrombin aptamer for being 1 μM by 100 μ L concentration GGG TGA CT-3 ' reacts 1 hour with 50 μ L samples to be detected in D-PBS buffer at 25 DEG C, obtains fibrin ferment four Aggressiveness;
The D-PBS pH of buffer is 7.4, and group becomes potassium chloride 2.67mmol/L;Dipotassium hydrogen phosphate 8.1mmol/L;Di(2-ethylhexyl)phosphate Hydrogen sodium 1.47mmol/L;Sodium chloride 138mmol/L;Magnesium chloride 5mmol/L.
3. the method according to claim 1, wherein the concrete operation method of step (2) are as follows:
Be added in the fibrin ferment tetramer 10 μ L concentration be 0.4mg/mL Fe-MIL-88A solution, concussion reaction 3 minutes.
4. the method according to claim 1, wherein the concrete operation method of step (3) are as follows:
The H that TMB, 10 μ L concentration that 100 μ L concentration are 10mM are 10mM is added in the mixed liquor obtained to step (2)2O2Solution with And 730 μ L pH 3.0 HAc-NaAc buffer, react 20 minutes at 40 DEG C.
5. the method according to claim 1, wherein the concrete operation method of step (4) are as follows:
The H that 50 μ L concentration are 2mM is added into mixed liquor after reaction2SO4Solution reads absorbance value at 451nm.
6. the method according to claim 1, wherein step (4) the standard fibrin ferment curve refers to standard After the thrombin solution of concentration carries out the processing of step (1)~(3), sulfuric acid solution is added, is surveyed and is inhaled using ultraviolet specrophotometer Luminosity, using corresponding concentration of thrombin as abscissa, with the concentration thrombin solution, absorbance is at ultra-violet absorption spectrum 451nm The curve that ordinate is drawn.
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CN110095420A (en) * 2019-05-08 2019-08-06 国家纳米科学中心 A kind of detection method and its application of concentration of hydrogen peroxide
CN110567953A (en) * 2019-10-12 2019-12-13 山西师范大学 Used for detecting Fe in environmental water sample and serum2+Content visual detection kit and detection method thereof
CN111504986A (en) * 2020-04-03 2020-08-07 上海理工大学 Method for rapidly detecting diamine biogenic amine

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CN110095420A (en) * 2019-05-08 2019-08-06 国家纳米科学中心 A kind of detection method and its application of concentration of hydrogen peroxide
CN110567953A (en) * 2019-10-12 2019-12-13 山西师范大学 Used for detecting Fe in environmental water sample and serum2+Content visual detection kit and detection method thereof
CN110567953B (en) * 2019-10-12 2022-07-01 山西师范大学 Used for detecting Fe in environmental water sample and serum2+Content visual detection kit and detection method thereof
CN111504986A (en) * 2020-04-03 2020-08-07 上海理工大学 Method for rapidly detecting diamine biogenic amine

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